Lowering Abeta levels and reducing amyloid deposition in the brain may be efficacious for Alzheimer's disease (AD). Abeta is the main component of amyloid plaques, and Abeta(42) correlates with AD. The beta-secretase, BACE1, is the enzyme that initiates Abeta generation and therefore it (and gamma-secretase) is a prime drug target for AD. The p3 peptide (Abeta17-40/42), the product alpha of gamma-secretase cleavage, is a major component of diffuse amyloid plaques that form early in AD. p3 is assumed to be non-amyloidogenic, however this has not been rigorously tested in vivo. We will study this question using BACE1 deficient mice, which have high levels of p3 and lack Abeta in the brain. In principle, the combined inhibition of both BACE1 and 7-secretase may more effectively reduce Abeta levels and amyloid deposition than inhibition of either enzyme alone. We will test this hypothesis by analyzing mice with compound BACE1 and presenilin heterozygous null mutations. Finally, the value of BACE1 as a drug target depends on whether BACE1 inhibition is free of toxic side effects, which in turn depends on the physiological functions of BACE1. To elucidate BACE1 functions, we will determine pathways and substrates of BACE1 using genomic and proteomic approaches. Our overall hypothesis is that BACE1 is a valid drug target for AD. The long-term goal of this project is to provide evidence that BACE1 inhibition is likely to be beneficial and have low toxicity for the treatment of AD. The Specific Aims are the following: 1. Determine whether p3 is amyloidogenic in vivo; 2. Determine whether combined genetic reduction of BACE1 and presenilin is more effective for decreasing Abeta production and amyloid deposition than reduction of either enzyme alone; 3. Identify BACE1 physiological functions and substrates using genomic and proteomic strategies. We will use transgenic and knockout mice and cultured primary neurons as model systems. Analytical approaches will include molecular (Northern, RTPCR, Western, immunoprecipitation, ELISA, gene microarray, 2D-PAGE, protein fingerprinting) and cellular (immunohistochemistry/light microscopy, immunofluoescence/confocal microscopy) techniques. These Aims will test our overall hypothesis that BACE1 is a valid drug target, and we expect our results will strengthen the conceptual foundation for the development of BACE1 and gamma-secretase inhibitors for AD.